The present invention relates to a method of identifying peptides useful for the treatment and monitoring of autoimmune diseases, especially arthritis, and to the peptides and proteins thus identified and their use in therapy and monitoring of the disease.
Autoimmune Diseases
One of the most intriguing characteristics of the immune system is its unlimited specificity. When threatened by potential dangerous foreign substances (antigens), including pathogens, the immune system mounts a tailor-made response. This tailor-made response is provided by the immune system""s antigen specific T and B lymphocytes. The virtually unlimited repertoire provided by these immune cells calls for a tight regulatory system preventing unwanted responses against our own (self) antigens. For years it was thought that the immune system was able to discriminate between self and non-self. However, with the growing knowledge of immunology, this theory has become more and more unsatisfactory. The self/non-self paradigm does not explain why perfectly healthy individuals can have circulating autoreactive T and B cells without any symptoms of autoimmune diseases.
Recently, a new concept providing more satisfactory explanations for the lack of autoimmune reactions in healthy individuals has been developed. In this new hypothesis, the decision whether the immune system is activated does not solely depend on the recognition of an antigen as foreign, but also on the immune system""s judgment whether it imposes danger to the integrity of the individual. The immune response must be considered as an outcome of a complex interaction between the lymphocyte and the antigen presenting cell (APC) in the context of cognate costimulatory signals, and the local cytokine microenvironment in which the recognition of the specific antigen takes place. This new view on the immune system does not only provide explanations for issues that made us doubt about the self/non-self paradigm, it also provides more insight in the mechanisms of central and peripheral tolerance. In the view of this new concept it is hypothesised that autoimmune diseases are the result of a qualitative or quantitative defect in the regulatory capacity of the immune system to control the naturally occurring autoreactive T-cell repertoire. It is therefore most important to develop novel therapeutic strategies for autoimmune diseases, such as rheumatoid arthritis, that aim at the reestablishment of such regulatory mechanisms of the immune system.
Rheumatoid Arthritis
Rheumatoid Arthritis (RA) is considered to be an autoimmune disease with chronic inflammation of the synovial membrane. RA is the most common inflammatory cause of disability in the western world. The prevalence of RA is approximately 1% of the population (range 0.3 to 2.1 percent); women are affected about three times more often than men. Specific characteristics of patients with RA are a variable degree of joint destruction and symmetric synovitis of their peripheral joints. Although the underlying cellular and molecular mechanisms have remained unclear until now, RA seems to provide a good example of how the interaction between genetic and environmental factors may lead to autoimmunity. Whilst environmental risk factors remain elusive, an association was found of RA with HLA-DR4 or a consensus sequence (QKRAA (SEQ ID NO: 1) or QRRAA (SEQ ID NO: 2)) in the hyper-variable region of the DRB1 molecule. Identification of the underlying cellular and molecular mechanisms leading to RA is complicated by the time of diagnosis. Since the moment of first symptoms is not known, by definition no patients with an early stage of the disease are available. When RA is diagnosed, it is a most destructive joint disease and its clinical course is characterised by involvement of the small joints of the hands and feet followed by centripetal progression to larger joints and finally even to the cervical spine. The histology of the disease is characterised by hyperplastic synovial tissue which is heavily infiltrated by various types of leucocytes. It is very likely that the constant supply of new cells of the immune system is necessary to induce and subsequently maintain the inflammatory process. Growing evidence for the involvement of T cells in RA is provided by the beneficial effect for RA patients from therapy that down regulates the effects of these lymphocytes.
The current treatments for RA are only symptomatic and can be divided into three lines. The first line of therapy consists of treatment with non-steroidal anti-inflammatory drugs (NSAIDs). These drugs can control pain and swelling of the joints, but do not halt the progressive joint destruction associated with the disease. Furthermore, NSAIDs can cause upper gastro-intestinal tract bleeding upon prolonged usage. Where RA remains active despite treatment with NSAIDs (which is usually the case), the second line of therapy can be applied, that consists of treatment with disease modifying anti-rheumatic drugs (DMARDs). These DMARDs, such as penicillamine, chloroquine, gold compounds and sulfasalazine, generally show some beneficial effect after a period of 3-6 months. However, due to severe side effects, treatment with DMARDs has to be stopped in about 25% of the patients. The belief that the immune system is actively involved in the onset and pathogenesis of RA has led to the development of a third line of treatment with strong broad-acting immunosuppressive drugs such as cyclosporin A and methotrexate. These strong immunosuppressive drugs generally have severe side effects, such as nephrotoxicity and in the long-term cancer. Besides the adverse side effects with the currently used anti-rheumatic drugs, the long-term outcome of sequential mono-therapy based on the therapeutic pyramid described above has been disappointing. No substantial evidence has been found proving that the currently used conventional drugs actually arrest the progression of joint destruction. Therefore, alternative strategies such as immunotherapy are clearly needed.
Optimal drugs for the treatment of autoimmune diseases such as RA would be able to attenuate the autoimmune process by re-establishing the immune system""s self-regulatory mechanisms that have failed and resulted in the autoimmune attack. Treatment during the early phase of the autoimmune process with such drugs have the potential to arrest the disease process. It has been demonstrated that T cells play a central role in the auto-destructive process in RA (Sigall et al., Clin. Exp. Rheum. 6:59 (1988)). Treatments that selectively suppress the activity of such autoreactive T cells can therefore be preferred. Such treatment could consist of the administration of an autoantigen or peptides derived thereof. This type of treatment has been very successful in the suppression of disease symptoms in various experimental autoimmune disease models in laboratory animals (Cremer et al., J. Immunol. 131: 2995 (1983); Myers et al., Immunol. 90: 161 (1997)). Oral or nasal administration of type II collagen before induction of collagen-induced arthritis can prevent disease induction in this mouse model. However, it is unclear at present time whether native type II collagen is really involved in the primary pathogenesis of RA. Thus the real autoantigens that are the target for autoreactive T cells in RA and that can be used for the generation of a therapeutic formulation for the treatment of RA may not have been identified. Despite the discussion about the role of native type II collagen in the primary pathogenesis of RA, the above described results in animal models have been translated into clinical testing in humans. Initial clinical studies have suggested clinical efficacy of oral toleration for RA with chicken type II collagen (Trentham et al., Science 261: 1727 (1993)). However, recent results of a large phase III clinical study have shown that oral treatment with type II collagen does not result in statistical significant clinical benefit for RA patients. Other proteins have been proposed as relevant target antigen in rheumatoid arthritis. One of these, human cartilage glycoprotein 39 (WO 96/13517), was recently described as potentially useful for the treatment of rheumatoid arthritis.
A major problem in the evaluation of the efficacy of novel immunotherapies aiming at T cell modulation, is the lack of proper tools to isolate and characterise T cells at the clonal level. The current development of multimeric MHC/peptide carrier systems to bind and isolate T cells in an antigen specific fashion, however opens novel possibilities for evaluating changes in T cell responses during and after immunotherapy (Kozono et al., Nature 369: 151 (1994)).
The invention relates to peptides and proteins suitable for the prophylaxis, treatment, diagnosis and/or monitoring of autoimmune diseases, including arthritis, said peptide comprising a contiguous sequence of 9 amino acid residues (nonapeptide), wherein the peptides have been selected by compliance with the amino acid sequence X1-X2-X3-X4-X5-X6-X7-X8-X9, wherein
X2, X6 and X7 are any amino acid,
X1 is any amino acid except K,H,R,E,D;
X3 is S or T;
X4 is one of F,L,I,V,A,G,C,P;
X5 is one of A,G,C,P,S,N,T,V;
X8 is one of V,L,I,M;
X9 is E or D.
For peptides suitable in relation to arthritic conditions, X1 is in particular A, F, G, I, L, N, P, Q, S, T, V, W, and X4 is in particular F, L, I, V, A, G, P. The peptide should preferably be derived from a mammalian cartilage, joint or arthritis-related protein such as collagen or a cartilage protein, and be capable of being recognised by freshly isolated T cells from rats in which Adjuvant Arthritis is induced and/or be capable of being recognised in vitro by T cell clone A2b. These peptides as such and proteins containing them are candidates for the prophylaxis, treatment, diagnosis and monitoring of arthritis-like conditions. If the rat sequence differs from the human sequence, the human homologue peptide/protein is selected as well for studies in RA and JCA patients. Suitably the isolated T cells are A2b-like.
In addition to to the nonamer sequence, the peptides according to the invention may comprise further amino acids derived from the relevant protein at either side of the nonamer, especially up to 11 further amino acids. Most preferably, the peptide contains 12-18 amino acids from the relevant (autoimmune-related) protein.
Peptides which contain mutations, especially conserved mutations, in the selected sequence are also part of the invention, and can be useful in arthritis treatment and monitoring. Peptides which contain at least five, especially at least seven amino acid residues which are in the same relative position as the corresponding amino acids of the sequences selected as above are also claimed.
The invention further relates to a method of selecting these peptides and to the use of the peptides and proteins containing these sequences in the prophylaxis and treatment of arthritis and arthritis-related conditions. The invention also provides a pharmaceutical composition containing one or more peptides or proteins as described above, optionally together with stabilisers, excipients, adjuvants or the like, in a pharmacologically acceptable carrier, for prevention or treatment of autoimmune diseases, including arthritic conditions.
The invention also pertains to such peptides and proteins for use in diagnosis and monitoring of arthritis, for example by using such natural or mutated peptides or proteins as antigens in an immunoassay of a biological sample wherein the presence of antibodies against or T cell recognition of such peptides and proteins is measured. The immunoassay may be of the competition type, or of the sandwich type or any other suitable type, such as e.g. the use of multivalent MHC/peptide carriers to isolate and characterise T cells. The proteins, peptides or carriers may or may not be labelled by conventional labels. The invention also provides a diagnostic kit containing at least a peptide or protein as described above, for detecting and monitoring arthitis or arthritis-like conditions. The invention is also directed at MHC-peptide complexes of the peptides according to the invention and at the use thereof for monitoring, diagnosing or treating arthritis or arthritis-like conditions.